Assessing sensitivity and reproducibility of RT-ddPCR and RT-qPCR for the quantification of SARS-CoV-2 in wastewater

Abstract

Throughout the COVID-19 global pandemic there has been significant interest and investment in using Wastewater-Based Epidemiology (WBE) for surveillance of viral pathogen presence and infections at the community level. There has been a push for widescale implementation of standardized protocols to quantify viral loads in a range of wastewater systems. To address concerns regarding sensitivity, limits of quantification, and large-scale reproducibility, a comparison of two similar workflows using RT-qPCR and RT-ddPCR was conducted. Sixty raw wastewater influent samples were acquired from nine distinct wastewater treatment plants (WWTP's) served by the Hampton Roads Sanitation District (HRSD, Virginia Beach, Virginia) over a 6-month period beginning March 9th, 2020. Common reagents, controls, master mixes and nucleic acid extracts were shared between two individual processing groups based out of HRSD and the UNC Chapel Hill Institute of Marine Sciences (IMS, Morehead City, North Carolina). Samples were analyzed in parallel using One-Step RT-qPCR and One-Step RT-ddPCR with Nucleocapsid Protein 2 (N2) specific primers and probe. Influent SARS-CoV-2 N2 concentrations steadily increased over time spanning a range from non-detectable to 2.13E + 05 copies/L. Systematic dilution of the extracts indicated that inhibitory components in the wastewater matrices did not significantly impede the detection of a positive N2 signal for either workflow. The RT-ddPCR workflow had a greater analytical sensitivity with a lower Limit of Detection (LOD) at 0.066 copies/μl of template compared to RT-qPCR with a calculated LOD of 12.0 copies/μL of template. Interlaboratory comparisons using non-parametric correlation analysis demonstrated that there was a strong, significant, positive correlation between split extracts when employing RT-ddPCR for analysis with a ρ value of 0.86.

Keywords: Molecular methods; PCR; SARS-CoV-2; Wastewater.

Graphical abstract

Fig. 1

Effect of Dilution on Detection of SARS-CoV-2 RNA in Wastewater: Extracts had been diluted at both a 1:2 (black dots) and 1:5 dilution (grey dots) in AE buffer prior to reverse transcription and PCR amplification and samples were analyzed using both (a) RT-ddPCR and (b) RT-qPCR by the processing group based out of IMS.

Fig. 2

Direct Comparison of Workflow Performance and Sensitivity: Sample extracts that had been diluted at a 1:2 dilution in AE buffer were analyzed in parallel using RT-ddPCR (black dots) and RT-qPCR (grey dots) by the processing group at IMS. N2 concentrations are presented as log transformed copy numbers per liter of influent filtered. A qualitative comparison between workflows resulted in a ĸ value of 0.314 with overall an overall agreement of 73.8%. Spearman rank correlation analysis resulted in a ρ value of 0.717.

Fig. 3

RT-ddPCR is the more Sensitive Workflow Regardless of WWTP Origin: Samples originating from the same WWTP were analyzed using the two previously described molecular workflows of RT-ddPCR and RT-qPCR. For all WWTPs with > than 7 collection events, RT-ddPCR was able to outperform RT-qPCR with respect to the detection of SARS-CoV-2 RNA.

Fig. 4

Interlaboratory Assessment of Reproducibility for RT-ddPCR Workflow: Nucleic acid extracts that had been shared between both HRSD (black dots) and IMS (grey dots) were both analyzed using the RT-ddPCR workflow in parallel. There was fair agreement between both groups based on kappa coefficient analysis with a ĸ value of 0.31 and a 95% positive percent positive agreement with a 73.8% overall percent agreement. Spearman correlation analysis provided a ρ of 0.86 which is evidence of a strong relationship between results produced by both groups.